In 2013, this project provides state-of-the-art research technologies for NIAID's intramural infectious diseases, allergy, and immunology research programs. The new technologies are developed and validated and then applied in support of NIAID research. Technologies developed outside the NIH are likewise tested, evaluated, validated and, if appropriate, incorporated into the technology portfolio of the NIAID intramural program. The technologies supported include flow cytometry, confocal microscopy, electron microscopy, DNA microarray, DNA sequencing, Next Generation sequencing, bacterial phenotyping and quantitative PCR. Many of these technologies are used in high containment laboratories critical to the Institute's infectious diseases and biodefense research agenda. In addition to technology development, the RTB provides advanced training in all aspects of the technologies in the Branch's portfolio. Sequencing:Capillary DNA sequencing uses the AB 3730XL with 96 well high throughput processing. Read lengths average >900 base pairs per read for vector and amplicon sequencing. Applications are developed in close collaboration with DIR investigators. All data is uploaded to a server, which tracks and manages all of the sequencing data generated for the Institute. Next Generation sequencing employs the Illumina GA II, the Illumina HiSeq 2000, and the 454 FLX Titanium FLX sequencers towards small RNA discovery, ChIP-Seq, transcriptomics, exome sequencing, de novo and ref-map genome sequencing, and copy number variation studies. Microarrays: The RTB develops project-specific research applications on Affymetrix microarray platform including custom chip design, experimental design, sample processing and chip processing. In addition to developing new applications for microarray research, the RTB develops statistical analysis, data management, and data mining solutions for DIR research programs; focusing on interpreting data generated by highly parallel detection systems used in genomics. The RTB also performs QPCR for high throughput microarray data validation, sample optimization, and Next gen data validation Human/Pathogen Genotyping: Several technologies are used for human and pathogen genotyping depending on the scope of the genotyping project and they range from capillary-based re-sequencing of entire human genes for de novo SNP or In/Del discovery, to high throughput targeted SNP genotyping via allelic discrimination assays using Taqman quantitative PCR, to SNPlex multiplexed assays (3730XL sequencer), to Affymetrix SNP chip-based arrays and custom pathogen SNP (MIP technology) arrays. Next Generation sequencing technologies also are playing a larger and developing role in de novo SNP, InDel, copy number, alternative splice variant and new expressed region discovery for both human and pathogen genomes. Bioinformatics data analysis is performed for Next Generation, microarrays and QPCR data analysis. High RAM, task-specific servers loaded with state-of-the-art software are used by skilled bioinformatisists towards providing DIR scientists with finished, publishable data sets where discoveries are clearly highlighted and presented for ease of interpretation. Data sets are stored in multiple formats for quick retrieval and re analysis and or comparative analysis with new or recently published competing datasets. Public data submissions and corrections to submissions are also performed by our Bioinformatisists. Flow Cytometry Project-specific research applications are developed for flow cytometry analysis and sorting in BSL-2 and BSL-3 environments. Electron Microscopy Project-specific research applications are developed in the areas of sample preparation and analysis ranging from basic structural studies to immune-localization of selected antigens for a wide array of specimens. A variety of methods, protocols, and equipment are employed to accommodate different preparative and imaging needs. Recent technological advancements have focused on two principle areas. One is the introduction and optimization of sophisticated preparative technologies and techniques for improved retention and visualization of labile structures often lost during routine processing and improving structural preservation. The other area involves the introduction of advanced imaging technologies including high resolution transmission and scanning electron microscopes. Finally, the Unit has been developing correlative techniques useful for examining transient or dynamic events by light microscopy to identify regions of interest which can then be prepared for visualization by electron microscopy to definitively correlate structures with functional assays. Conventional specimen processing for examination by electron microscopy requires use of chemicals that often extract or alter structures of interest. Cryo-preservation through high pressure freezing followed by chemical exchange at low temperature in a process called freeze substitution has become the preferred technique allowing retention of fragile structures. Freeze substitution is the lengthy process of replacing vitreous ice in rapidly-frozen hydrated samples with an organic solvent containing fixatives and electron-dense contrasting agents. In order to determine whether controlled microwave irradiation could facilitate the process, the Electron Microscopy Unit developed and assessed methods for maintaining cryo conditions in a laboratory microwave processor. Further development resulted in the fabrication of a thermally controllable unit decreasing processing periods from several days to a few hours while achieving excellent structural and antigenic preservation. The Microwave Assisted Freeze Substitiution concept resulted in a patent application. The 300 kV TEM microscope recently acquired by the EM Unit is the most advanced instrument for high resolution 3-D biological imaging configured to have optimal flexibility to respond to the needs of multiple investigators and provide them with the highest quality images available with extant technology. It is the platform upon which improvements in ultrastructural imaging will likely be made over the next decade resulting in the highest level resolution possible for the characterization of macromolecular complexes, cellular organelles, viruses, bacteria and other parasites as well as the ability to observe in three dimensions the host pathogen interactions occurring within eukaryotic cells. These technologies improve our ability to relate structure to function, providing information which may identify vaccine targets or other intervention strategies. Current projects include high resolution imaging and reconstruction of bacteria, viruses, macromolecular complexes and eukaryotic cells. The ability to assign cellular function to structures provides valuable information in the study of host pathogen interactions. Fluorescent labeling antigens of interest or use of green and red fluorescent proteins as genetic markers allows visualization of transient and dynamic events by light microscopy (LM). Although advances in technology have improved resolution by LM, electron microscopy (EM) still provides superior ability for resolving small structures. EM however provides only a snapshot since specimens are immobilized during specimen processing, limiting information about cellular dynamics. Correlative light and electron microscopy (CLEM) bridges this gap by coupling dynamic or transient information available through LM with the ability to resolve ultrastructural details by EM. Innovative methods have been developed by the Unit that enable identification and imaging cells of interest first by confocal laser or epifluorescent microscopy, and then by scanning or transmission electron microscopy (SEM & TEM respectively.)